Seeing Cellular Excitation

Seeing Cellular
Excitation

Whether for basic research or drug discovery, precise measurement of voltage changes at the cell membrane is essential for understanding function, pathology, and potential therapeutic effects in electrically active cells.

The traditional "gold-standard" method of patch clamping provides single-cell data with outstanding sensitivity and time resolution. Though very precise, patch clamping is also labor intensive and slow, and not well suited to multi-cell and longitudinal studies.3,8,9,10,12

Try this decision tree for the right frame rate for visualizing five types of time-sensitive experiments using the ORCA-Flash4.0 camera—read now.

Above: Still image of a spiral wave, which serves as a stable source for periodic electrical spiking.

Above: Spiral wave center. The fluorescence change is plotted in color on a dark red (low voltage) to yellow (high voltage) color map. The induced fluorescence change is overlaid on a grayscale image of the average fluorescence level. Field-of-view is 400x400 microns, playback slowed five-fold. Video courtesy of Christopher Werley.

However, capturing transient, millisecond events such as action potentials requires an indicator that responds very quickly to voltage changes. A new experimental method from Park, et al.6 of Adam Cohen's lab at Harvard University makes it possible to screen candidate indicator molecules for response times down to 4 ms, while also checking for the bright signal and efficient localization to the plasma membrane needed.

THE BARRIERS

Patch-clamp recording uses an electrode inside a micropipette to measure electrical potential across the cell membrane, in whole cells or even individual ion channels, thanks to a high-resistance seal between the pipette tip and cell membrane. Whole-cell methods used to monitor action potentials provide precise quantitation, beyond the limits of the current optical assay, but take much more time and manual effort.2,6,12

Optical methods allow high-throughput observation of activity from the subcellular level to multicellular networks. However, they have been limited by the challenge of developing voltage-sensitive indicators that are free from toxicity and pharmacologic effects, localized to the plasma membrane specifically, and highly efficient in producing a bright signal in response to voltage changes—within milliseconds.3,6

The ORCA-Flash4.0 scientific CMOS camera's wide field of view, fast frame rates and high effective quantum efficiency enabled live cell video of action potentials using fluorescence. But what if luminescent markers are needed? Compare sCMOS and EM-CCD technology to find the right camera for the job—read more.

THE SOLUTION

Above: Plane wave propagation. A video of voltage recorded using the voltage sensitive dye VF2.1.Cl showing a plane wave, the most common electrical propagation pattern. Brightness is proportional to voltage-induced change in fluorescence. The field of view is 3x6 mm and the playback is slowed five-fold from real time. Image courtesy of Christopher Werley.

Park, et al., demonstrated the sensitivity and responsiveness of the new assay by confirming its results against published attributes for several well-characterized dyes and indicator proteins, as well as patch-clamp studies of their own. Then they tested the assay in screening for candidate genetically expressed protein indicators, using mutant strains of bacterial rhodopsin, called Archeorhodopsin. With excellent sensitivity and speed, Archeorhodopsin has been used to resolve individual action potentials in cultured neurons.5 Park and colleagues identified several new variants with even better speed and sensitivity.

Video microscopy of single cells at 60x showed a wavefront of action potentials crossing the HEK cell monolayer. The assay showed good temporal resolution, even at single-cell resolution. The depolarization wave took about 1 ms to cross a cell, with the voltage rise taking about 3 ms. The mean action potential rise time was 2.5 ms +/- 1.3 ms.

To get the apparent response time of each candidate indicator, the researchers convolved camera exposure time (~1 ms), cellular voltage rise time (~2.5 ms) and true response time of known indicator. Based on that, the authors estimate that the new method can measure response times down to and below about 4 ms. That's faster than most transgenic voltage indicators in the literature so far, making this assay an excellent tool to filter candidates for the fastest fluorescent voltage indicators.

THE POSSIBILITIES

“… intracellular recordings of the full electrophysiological repertoire … are, at present, obtained only by sharp or patch microelectrodes. These, however, are limited to single cells at a time and for short durations.”

SPIRA, ET AL.8

“Voltage imaging, on the other hand, could in principle capture the entire picture: reading out the electrical activity of each neuron in the circuit, including sub-threshold excitatory and inhibitory events, for all different cell types.”

PETERKA, ET AL.3

“Patch-clamp measurements in a fluorescence microscope provide quantitative and precise data, but manual patch clamp is laborious and slow.”

PETERKA, ET AL.6

“… these probes allow us to measure and control neuronal signals with spatial resolution and genetic specificity that already greatly surpass those of electrophysiology.”

PARK, ET AL.6

“Optical recording with a voltage-sensitive dye is advantageous where membrane potential must be recorded in many sites at once.”

ZECEVIC, ET AL.11

Park and colleagues’ new platform can quantify fluorescent marker response times of between about 4 and 50 ms, and provides both high speed and high sensitivity visualization of electrophysiological events. Validated on fluorescent voltage-sensitive dyes and genetically expressed indicators, this assay could be used to test any type of fluorescent voltage indicator. The HEK cell model could also prove useful for basic research into cellular electrical activity and drug discovery, observing candidate drug effects on the spiking waveform and propagation.